West Nile Virus (WNV) is an emerging mosquito-borne human and animal pathogen (NIAID Category B) that has caused outbreaks of fatal encephalitis in Europe, Asia, the Middle East, and most recently, the United States. Despite the increasing numbers of cases and deaths attributed to WNV, no effective therapy or vaccine is available for humans. Recently, using a mouse model of WNV encephalitis, we have demonstrated that a deficiency of antibody leads to disseminated infection, and passive administration of immune serum or purified polyclonal antibody protects antibody-deficient and wild type mice from WNV-induced mortality. Based on these observations, the proposed research plans to generate a new panel of monoclonal antibodies (mAbs) against WNV E and NS1 proteins, test their therapeutic efficacy in our mouse model of WNV encephalitis, and humanize the best candidates for possible clinical intervention. In Specific Aim 1, a panel of mAbs against the E and NSl proteins of WNV will be generated and characterized for neutralizing activity and effector function, and for localization to discrete structural domains on each protein. In Specific Aim 2, the mAbs with the greatest inhibitory and effector activity in vitro will be evaluated for therapeutic efficacy in the mouse model of WNV infection. Because the RNA-dependent RNA polymerase of WNV has a high error rate and thus, a potential to rapidly alter immunodominant residues, trials will be also conducted with combinations of mAbs. A novel ranking system has been developed to streamline the selection process of mAbs with the greatest potential inhibitory activity. In Specific Aim 3, the six anti-E and anti-NS1 mAbs with the greatest inhibitory activity will be humanized using chimerization and complementarily determining region (CDR) grafting techniques and re-evaluated in vitro and in vivo for anti-WNV activity. By studying the mechanisms of antibody-mediated protection, we hope to rationally design immunotherapeutic agents against WNV. These studies are an essential first step in the generation of humanized mAbs that have utility as therapeutic agents against WNV in humans.